Antiviral and anti-inflammatory activities of chemical constituents from twigs of Mosla chinensis Maxim

Seven undescribed compounds, including three flavones (1–3), one phenylpropanoid (19), three monoaromatic hydrocarbons (27–29), were isolated from the twigs of Mosla chinensis Maxim together with twenty-eight known compounds. The structures were characterized by HRESIMS, 1D and 2D NMR, and ECD spectroscopic techniques. Compound 20 displayed the most significant activity against A/WSN/33/2009 (H1N1) virus (IC50 = 20.47 μM) compared to the positive control oseltamivir (IC50 = 6.85 µM). Further research on the anti-influenza mechanism showed that compound 20 could bind to H1N1 virus surface antigen HA1 and inhibit the early attachment stage of the virus. Furthermore, compounds 9, 22, 23, and 25 displayed moderate inhibitory effects on the NO expression in LPS inducing Raw 264.7 cells with IC50 values of 22.78, 20.47, 27.66, and 30.14 µM, respectively. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-024-00448-w.


Graphical Abstract 1 Introduction
Influenza viruses had high pathogenicity and infectiousness, and is an important risk factor for human health.It had been exhibited the ability to invade the epithelial cells of the respiratory tract for the happening of the inflammation, and thereby result in influenza with the symptom such as fever, headache, and muscle pain.Influenza is one of the most common respiratory diseases.If the patients had not effective medical interventions, it could induce serious complications such as pneumonia, acute lung injury and even pulmonary fibrosis [1,2].Influenza viruses induced diseases had been become a worldwide public health problem and the main treatment is vaccine or drug.However, because of the extraordinary high rate of virus mutation and the side effects of existing drugs, it's essential to find ingredients with high effect and low toxicity from natural food.Phenolic compounds containing multiple phenolic hydroxyl groups, which can bind with targeting proteins of disease and possess significant activities of antioxidant, antiviral, and anti-inflammatory.

Results and discussion
The 1 H and 13 C NMR spectroscopic data of 1 (Table 1) is highly similar to acacetin [40], except for the additional presence of sugar moiety in 1.The above observation indicated 1 was glycoside derivative of acacetin.The position of glycosyl junction was identified by HMBC map.A series of HMBC correlations from H Xyl 1) were highly analogue to those of 1 except the presence of two acetyl groups at δ C 170.5, 170.7, 20.7, and 21.1 in 2, as well as the minor change of chemical shifts in two sugars.Hence, 2 was deduced to be the acylated derivative of 1. Acid hydrolysis demonstrated the glycosidic nature of 2, which was identified as the β-D-glucose and β-D-apiose by the GC analysis and coupling constant values (J H-1ʹʹ, H-2ʹʹ ) (Additional file 1: Fig. S61).The position of glycosyl junction was identified by the HMBC correlations from H Glc -1 (δ H 5.25) to C-7 (δ C 163.0), from H Api -1 (δ H 5.29) to C Glc -2 (δ C 76.9), from H Glc -2 (δ H 4.04) to C Api -1 (δ C 108.5).Furthermore, the sequence of the acetyl groups was deduced to be connected to C Glc -6 and C Api -5 due to the HMBC correlations from H Glc -6 (δ H 3.79/3.20)to C-7ʹʹ (δ C 170.5), from H Api -5 (δ H 3.66/3.52)to C-6ʹʹʹ (δ C 170.7).Therefore, compound 2 was identified as acacetin 7 1).

Biological evaluation 2.2.1 Anti-influenza A virus activity
The activity of compounds (6-18, 20-26, [31][32][33][34][35] against the influenza virus was evaluated by using A/ WSN/33/2009 (H1N1) infected MDCK cells.In comparison with the positive control oseltamivir with IC 50 = 6.85 µM, compound 20 exhibited significant inhibition effects of H1N1 (IC 50 = 20.47 µM); However, other compounds had no anti-influenza activity.The results of western blot analysis showed that 20 could dramatically reduce the nucleoprotein protein expression at 2, 5, and 8 h, indicating that 20 inhibits influenza virus infection by interfering with the beginning phase in the viral life cycle (Fig. 6).Furthermore, the nucleoprotein distribution in infected cells was observed by fluorescence microscopy (Fig. 7).It showed that after virus infection for 2 and 5 h, the virus population in the MDCK cells of the DMSO group was dramatically higher than that of the experimental group.This result further indicated that the influenza virus could be inhibited by compound 20.-6 , and red blood cells mixed with virus could not agglutinate.Normal red blood cells produce cell agglutination at room temperature.B The compound could effectively promote erythrocyte agglutination at 40 μΜ and 80 μΜ, the compound had no effect on red blood cells.C The HA1 polypeptide is colored purple, HA2 is green, and 20 is yellow.20 can bind with HA1 residues Glycoprotein hemagglutinin (HA) of the influenza virus has been used as a potentially important target for developing anti-influenza drugs [45].A hemagglutinin inhibition (HI) assay was designed to check if 20 could prevent virus attachment to the cells through disturbing the connection between HA and cellular receptors.The results showed that 20 might effectively promote erythrocyte agglutination at 40 μM (Fig. 8A and B).The results indicated that 20 could bind to influenza virus surface antigen HA1, inhibiting the early adsorption process of WSN.In addition, it was revealed that the docking sites with high-affinity underlying interaction could be intently related to residues ASN 68 and ARG 224 through Molecular docking (Fig. 8C).However, no binding sites between 20 and HA were observed on the receptor binding domain (RBD) of HA1 sialic acids.Therefore, it could be concluded that 20 exhibited the antiviral influence on A/WSN/33/2009 (H1N1) virus by targeting the hemagglutinin fusion machinery.

Anti-inflammatory activity
The influenza virus can lead to an excessive immune response and induce the production of inflammatory cytokines, such as IL-1 and IL-6 [46].So, it would be valuable if the drugs had both antiviral and anti-inflammatory activities.Hence, we used the cells (LPS-activated RAW 264.7) to evaluate the impact of compounds ( 6 3).

Plant material
The M. chinensis twigs were harvested in August 2019 from Honghe Hani and Yi Autonomous Prefecture (Yunnan, China), with the authentication of Dr. Jindong Zhong (Kunming University of Science and Technology).A voucher specimen (serial number: KMUST201903) was stored at the Department of Life Science and Technology.

Extraction and purification
The air-dried powdered of M. chinensis twigs (15 kg) was extracted with 70% acetone/H 2 O by refluxing for 24 h (30 L × 3 times).After filtration and evaporation procedures, the extract (1286 g) was yielded and thoroughly dissolved in H 2 O.The mixture was then extracted by petroleum ether, chloroform, ethyl acetate, and n-butanol, respectively.The ethyl acetate extract (145 g) was separated to four fractions (Fr.A-D) by silica gel column (20 × 300 cm) eluted with dichloromethane-methanol (1:0-0:1).

Acid hydrolysis and determination of the absolute configuration of sugars
Based on the method reported by Wu et al. [47], the D glucopyranose configurations in compounds 1-3 and 27 were measured.Compounds 1-3 and 27 (1 mg per compound) were individually mixed with 3 ml HCl (2 M).Each mixture was boiled for 4 h at 100 ℃.After neutralization with NaHCO 3 , the mixture was treated by EtOAc.Subsequently, the H 2 O layer was evaporated and dissolved in DMSO (1.0 ml) before the acetic anhydride (40 μL) and 1-Methylimidazole (20 μL).After the reaction, extracted with EtOAc and analysed by GC.Monosaccharide compositions in compounds were identified by coeluting with authentic monosaccharide.

ECD quantification
The conformational structures of compounds were achieved from Chem3D modeling and ROESY spectra.In terms of the conformations, low-energy conformers of 27 were created via CONFLEX software by using an energy window (10 kcal/mol, MMFF94S) [48].Density functional theory (DFT) method was employed to optimize the selected conformers in MeOH at the B3LYP/6-31 G (d) level [49].Geometry optimizations and predictions of the conformersʹ ECD spectra were conducted by TD-DFT-B3LYP/6-311G (2d, p) level using a solvent (IEFPCM solvent model for methanol) [50].SpecDis 1.71 was hired to generate the predicted curves of ECD, and Gaussian 16 package was applied to all predictions [51].After UV correction, compound 27 spectrum was weighted using the Boltzmann distribution.

Anti-influenza virus assay
Based on the approach described by Dang et al. [52], anti-influenza virus assay was carried out.Briefly, prior to infection, MDCK cells (8 × 10 3 cells/well) were cultivated for 24 h in 96-well plates, and the medium was removed.The mixture of compounds (at 3.125, 6.25, 12.5, 25, and 50 μM) and H1N1 virus was cultured at ambient temperature for 15 min and then transferred to the plates containing MDCK cells.The plates were stored at 37 °C with 5% CO 2 for 48 h.Subsequently, the antiviral activity was quantified using microscopy.The obtained antiviral activity was verified using the CellTiter-Glo luminescent cell viability assay (Promega, #G7570).Each compound's cytotoxicity was evaluated through incubating with uninfected MDCK cells for 48 h [53,54].

Immunofluorescence assay
MDCK cells (1 × 10 5 cells) were loaded into each well of 24-well plates.The plates were kept under 5% CO 2 condition at 37 °C.As the cells increased by 50%, the cells were added and cultured with A/WSN/33/2009 (H1N1) virus (MOI = 0.1) for 2 h.After the removal of the supernatant, the cells were rinsed two times using PBS.Subsequently, the 20 was supplemented to cells and stored under conditions of 5% CO 2 and 37 °C.At 2, 5, 8, and 10 h of incubation, the cells were fixed using PFA in PBS (4%, Beyotime Biotechnology) at refrigerated temperature for 10 min.The fixed cells were then permeabilized for 10 min at room temperature using 0.1% Triton X-100 in PBS and blocked for 1 h at 37 °C using 3% BSA in PBS.Afterward, the treated cells were first stored at 4°C overnight supplemented with nucleoprotein antibody diluted in 3% BSA (1:250, Abcam, CA, USA) and then cultured at ambient temperature for 1 h with fluorescein isothiocyanate (FITC)-labeled secondary antibody diluted in 3% BSA (1:250).The nucleus in cells was stained by DAPI for 10 min at ambient temperature.After staining, the fluorescence was examined by an inverted fluorescence microscope (Nikon A1R/ A1, Shanghai, Japan) [56].

Table 3
Inhibitory effect of 9, 22, 23 and 25 on LSP-induced NO production in macrophages a L-NMMA was used as positive control 377.1592 [M + H] + (calcd for C 20 H 24 O 7 , 377.1595); 1 H and